The rapid entry of calcium into cells is mediated by a class of proteins called voltage-gated calcium channels. Calcium channels are a heterogeneous class of molecules that respond to depolarization by opening a calcium-selective pore through the plasma membrane. The entry of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression. In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Miller, R. J. (1987) “Multiple calcium channels and neuronal function.” Science 235:46-52. Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitter. Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and has been implicated in long-term changes in neuronal activity.
In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. Recently, mutations identified in human and mouse calcium channel genes have been found to account for several disorders including, familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. Fletcher, et al. (1996) “Absence epilepsy in tottering mutant mice is associated with calcium channel defects.” Cell 87:607-617; Burgess, et al. (1997) “Mutation of the Ca2+ channel β subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse.” Cell 88:385-392; Ophoff, et al. (1996) “Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.” Cell 87:543-552; Zhuchenko, O. et al. (1997) “Autosomal dominant cerebellar ataxia (SCA6) associated with the small polyglutamine expansions in the α1A-voltage-dependent calcium channel.” Nature Genetics 15:62-69.
The clinical treatment of some disorders have been aided by the development of therapeutic calcium channel antagonists. Janis, et al. (1991) in Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance. CRC Press, London.
Native calcium channels have been classified by their electrophysiological and pharmacological properties as T, L, N, P and Q types (for reviews see McCleskey, et al. (1991) “Functional properties of voltage-dependent calcium channels.” Curr. Topics Membr. 39: 295-326, and Dunlap, et al. (1995) “Exocytotic Ca2+ channels in mammalian central neurons.” Trends Neurosci. 18:89-98.). T-type (or low voltage-activated) channels describe a broad class of molecules that activate at negative potentials and are highly sensitive to changes in resting potential. The L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them. L-type channels are sensitive to dihydropyridine (DHP) agonists and antagonists, N-type channels are blocked by the Conus geographus peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta. A fourth type of high voltage-activated Ca channel (Q-type) has been described, although whether the Q- and P-type channels are distinct molecular entities is controversial (Sather et al. (1993) “Distinctive biophysical and pharmacological properties of class A (B1) calcium channel α1 subunits.” Neuron 11:291-303; Stea, et al. (1994) “Localization and functional properties of a rat brain α1A calcium channel reflect similarities to neuronal Q- and P-type channels.” Proc Natl Acad Sci (USA) 91: 10576-10580.). Several types of calcium conductances do not fall neatly into any of the above categories and there is variability of properties even within a category suggesting that additional calcium channels subtypes remain to be classified.
Biochemical analyses show that neuronal high-threshold calcium channels are heterooligomeric complexes consisting of three distinct subunits (α1, α2δ and β)(reviewed by De Waard, et al. (1997) in Ion Channels, Volume 4, edited by Narahashi, T. Plenum Press, New York). The α1 subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel antagonists. The mainly extracellular α2 is disulphide-linked to the transmembrane δ subunit and both are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of brining to a cytoplasmic region of the α1 subunit. A fourth subunit, γ, is unique to L-type Ca channels expressed in skeletal muscle T-tubules. The isolation and characterization of γ-subunit-encoding cDNA is described in U.S. Pat. No. 5,386,025 which is incorporated herein by reference.
Molecular cloning has revealed the cDNA and corresponding amino acid sequences of six different types of α1 subunits (α1A, α1B, α1C, α1D, α1E and α1S) and four types of β subunits (β1, β2, β3 and β4)(reviewed in Stea, A., Soong, T. W. and Snutch, T. P. (1994) “Voltage-gated calcium channels.” in Handbook of Receptors and Channels. Edited by R. A. North, CRC Press.) PCT Patent Publication WO 95/04144, which is incorporated herein by reference, discloses the sequence and expression of α1E calcium channel subunits.
The different classes of α1 and β subunits have been identified in different animals including, rat, rabbit and human and share a significant degree of amino acid conservation across species (for examples see: Castellano, et al. (1993) “Cloning and expression of a third calcium channel β subunit.” J. Biol. Chem. 268: 3450-3455; Castellano, et al. (1993) “Cloning and expression of a neuronal calcium channel β subunit.” J. Biol. Chem. 268: 12359-12366; Dubel, et al. (1992). “Molecular cloning of the α1 subunit of an ω-conotoxin-sensitive calcium channel.” Proc. Natl. Acad. Sci. (USA) 89: 5058-5062; Fujita, et al. (1993) “Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain.” Neuron 10: 585-598; Mikami, et al. (1989). “Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel.” Nature 340: 230-233; Mori, et al. (1991) “Primary structure and functional expression from complementary DNA of a brain calcium channel.” Nature 350: 398-402; Perez-Reyes, et al. (1992). “Cloning and expression of a cardiac/brain β subunit of the L-type calcium channel.” J. Biol. Chem. 267: 1792-1797; Pragnell, et al. (1991). “Cloning and tissue-specific expression of the brain calcium channel β-subunit.” FEBS Lett. 291: 253-258; Snutch, et al. (1991) “Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS.” Neuron 7: 45-57; Soong, et al. (1993) “Structure and functional expression of a member of the low voltage-activated calcium channel family.” Science 260: 1133-1136; Tomlinson, et al. (1993) “Functional properties of a neuronal class C L-type channel.” Neuropharmacology 32: 1117-1126; Williams, et al. (1992) “Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium channel subtype.” Neuron 8: 71-84; Williams et al. (1992) “Structure and functional expression of an ω-conotoxin-sensitive human N-type calcium channel.” Science 257: 389-395.
In some expression systems the α1 subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits. Until recently, the reported modulatory affects of β subunit coexpression were to mainly alter kinetic and voltage-dependent properties. More recently it has been shown that β subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, et al. (1994) “Voltage-dependent facilitation of a neuronal α1C L-type calcium channel.” EMBO J. 13: 5032-5039; Stea, et al. (1995) “Determinants of PKC-dependent modulation of a family of nuronal calcium channels.” Neuron 15:929-940; Bourinet, et al. (1996) “Determinants of the G-protein-dependent opioid mdoulation of neuronal calcium channels.” Proc. Natl. Acad. Sci. (USA) 93: 1486-1491.)
The electrophysiological and pharmacological properties of the calcium channels cloned to date can be summarized as shown in Table 1. While the cloned α1 subunits identified to date correspond to several of the calcium channels found in cells, they do not account for all types of calcium conductances described in native cells. For example, they do not account for the various properties described for the heterogenous family described as T-type calcium channels. Furthermore, they do not account for novel calcium channels described in cerebellar granule cells or other types of cells. (Forti, et al (1993) “Functional diversity of L-type calcium channels in rat cerebellar neurons.” Neuron 10: 437-450; Tottene, et al. (1996). “Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons.” J. Neurosci. 16: 6353-6363).
Because of the importance of calcium channels in cellular metabolism and human disease, it would be desirable to identify the remaining classes of α1 subunits, and to develop expression systems for these subunits which would permit the study and characterization of these calcium channels, including the study of pharmacological modulators of calcium channel function. Thus, it is an object of the present invention to provide heretofor undisclosed calcium channels having novel α1 subunits, including cell lines expressing these
TABLE 1ω-conotoxin1,4-ω-agatoxinω-conotoxinnativeGVIAdihydropyridinescadmiumIVAMVIICCa2+ channel typeα1A——✓✓✓P/Q-typeα1B✓—✓—✓N-typeα1C—✓✓——L-typeα1D—✓✓——L-typeα1E——✓——novelα1S—✓✓——L-typenew calcium channels. It is a further object of the present invention to provide a method for testing these novel calcium channels using such cell lines.